The MIP family of water-selective transmembrane pore proteins, commonly known as "aquaporins", are responsible for the rapid movement of water across cell membranes, and are thus crucial for solute transport and cellular, tissue, and organismal osmoregulation. Molecular defects in these proteins can result in cataract formation and problems in urine concentration. An exciting feature of several aquaporins is that their water permeability is actively gated. Water channel activity of AQP0 (formerly known as MIP), found in eye lens fiber cells, is regulated by both pH and calcium. Water channel activity of AQP4, found in brain neocortex and ocular ciliary bodies, is regulated by phosphorylation. Water channel activity of AQP6, found in renal collecting duct cells, is also regulated by pH. We hypothesize that electrostatic changes, resulting from either phosphorylation or amino acid charging, drive the allosteric pore modifications responsible for regulation. High resolution structural data of the closed and open states are essential to test this hypothesis. We previously grew well-ordered helical crystals of the aquaporin, alpha-TIP, which is gated by phosphorylation. A projection map at 7.7Angstroms, resolution showed conservation in the alpha-helical design of AQP0, AQP1 and alpha-TIP. Consequently, our structural studies on alpha-TIP serve as a paradigm for understanding gating mechanisms in aquaporins such as AQP0, AQP4, and AQP6. To explore the molecular basis of phosphorylation-dependent gating of the aquaporin alpha-TIP, we will pursue the following 5 specific aims: Aim 1: Use mass spectrometry to determine the phosphorylation sites of alpha-TIP. Aim 2: Use electron cryo-microscopy and image analysis to delineate the alpha-helical movements associated with phosphorylation-dependent gating. Aim 3: Develop an overexpression system to produce mutant aquaporins for site-directed cysteine spin labeling. Aim 4: Develop a functional assay for water channel activity of the mutant aquaporins. Aim 5: Use electron paramagnetic resonance (EPR) spectroscopy of spin-labeled aquaporins to define the amino acid rearrangements that regulate water transport. For this revised application, we have successfully expressed His(10)-tagged alpha-TIP in the methylotrophic yeast Pichia pastoris and used Ni-affinity chromatography to purify milligram quantities of the protein. In addition, we have developed an osmotic-shock assay in yeast spheroplasts to verify that the recombinant protein exhibits water channel activity. The structural details revealed by our analysis will be the first molecular description of phosphorylation-dependent channel gating. In addition, this project will provide insight into the molecular basis for channel regulation and will contribute to the general principles that govern conformational changes of membrane proteins.